Project Summary/Abstract. Tobacco addiction is the second leading cause of death in the world and the single largest cause of cancer and heart disease. Given present smoking trends, tobacco use will kill 10 million people each year by 2020. Each year nearly half of the 42 million adult smokers attempt to quit; yet, due to the highly addictive nature of nicotine, less than 5% succeed. Aids to smoking cessation include supportive counseling, nicotine replacement and receptor antagonists that reduce nicotine reward and withdrawal symptoms. Unfortunately, long-term outcomes for nicotine replacement therapies remain poor and achieve an abstinence rate of only 10-20% after the first year. Drugs of abuse vaccines induce antibodies that block the pharmacological effects of drugs like nicotine. To date, vaccines for smoking cessation have shown promise in preclinical animal models for their ability to diminish nicotine-mediated physiological and behavioral responses. However, in clinical studies these vaccines failed to measure significant differences in smoking abstinence between the intervention and placebo groups. The Achilles' heel of nicotine vaccines has been their inability to consistently generate high antibody concentrations to reduce circulating free drug. A biologic capable of catabolizing nicotine, rather than simply sequestering the drug, could overcome the shortcomings of a nicotine vaccine. The proposed study details a bacterial strain, Pseudomonas putida, which has evolved to use nicotine as its sole source of carbon and nitrogen. From this bacterial strain, was isolated a first-in-class enzyme, a nicotine oxidoreductase (NicA2), that is highly efficient at degrading nicotine to a non-psychoactive product. Characterization of the enzyme indicates it to be an excellent candidate for nicotine cessation therapy. Yet, successful demonstration of such a strategy will require improvement of the enzyme as outlined in specific aims including: (1) Engineering NicA2 to increase its stability in serum and reduce immunogenicity liabilities. (2) Evaluating the effect of NicA2 on the reinforcing properties of nicotine in non-dependent and dependent rodent models with high predictive validity for tobacco use disorder. My proposal will take an existing enzymatic strategy for the attenuation of nicotine's psychoactive effects and engineer a clinically viable therapeutic for nicotine cessation.